Preparation of Human Kidney Progenitor Cultures and Their Differentiation into Podocytes

Kidney diseases are a global health concern. Modeling of kidney disease for translational research is often challenging because of species specificities or the postmitotic status of kidney epithelial cells that make primary cultures, for example podocytes. Here, we report a protocol for preparing primary cultures of podocytes based on the isolation and in vitro propagation of immature kidney progenitor cells subsequently differentiated into mature podocytes. This protocol can be useful for studying physiology and pathophysiology of human kidney progenitors and to obtain differentiated podocytes for modeling podocytopathies and other kidney disorders involving podocytes.


A. Human kidney progenitor cells (KPC): isolation, maintenance, and cryopreservation
In this section, we describe how to isolate primary kidney progenitor cells from human tissue.The method we describe exploits the ~50-fold higher proliferative capacity of KPC cells in comparison to other renal cell types in a specific growth medium (Peired et al., 2020).Based on our experience, this method allows to obtain a pure population of viable kidney progenitors more easily than the multi-step process based on separation using magnetic beads.1. Isolation of KPC from human kidney tissue a. Collect a fragment of kidney cortex (from 1 to 3 cm 3 ) from the pole opposite to the tumor.Store the tissue in sterile physiological saline solution during transport to the laboratory.We recommend performing the kidney cell isolation within 1 h after surgical tissue collection.b.Remove the kidney capsule and transfer the sample to a 100 mm sterile dish.Mince the cortex in pieces as small as possible using a scalpel.Add to the dish 5 mL of 750 U/mL collagenase type IV prepared in EBM medium.Incubate for 45 min at 37 °C in the incubator.Neutralize the enzymatic reaction by adding 10 mL of EBM containing 10 % FBS.CAUTION: During mincing, maintain the tissue fragments humidified by adding a drop of sterile physiological saline solution.c.Transfer the suspension to graded mesh screens (60 and 80 mesh).Mechanically break down the tissue suspension using a glass pestle and pass it through the 60 and 80 mesh screens.Wash thoroughly with 20 mL of kidney progenitor cell washing medium and recover the flowthrough in a polypropylene urine container.Transfer this suspension to a 50 mL polypropylene tube and centrifuge at 400× g for 5 min at 4 °C.d.Aspirate the supernatant and wash the pellet with 5 mL of PBS.Centrifuge at 400× g for 5 min at 4 °C.Discard the supernatant and resuspend the pellet in 5 mL of red blood cell lysis buffer.e. Incubate for 4 min at 37 °C and then stop the reaction by adding 10 mL of kidney progenitor cell washing medium.f.Centrifuge at 400× g for 5 min at 4 °C.g.Remove supernatant and resuspend the pellet in 10 mL of kidney progenitor cell growth medium.h.Count cell suspension using a Bürker counting chamber.i. Transfer the cells in 75 cm 2 flask (500,000 cells/flask) in 8 mL/flask kidney progenitor cell growth medium.Label as passage 0. j.Place the cells in a 5% CO2, 37 °C incubator.k.After three days, replace the medium with fresh kidney progenitor cell growth medium to remove unattached cells and continue with twice-a-week changes until cells reach 80% confluency.It usually takes 7-10 days.2. Expansion and sub-culturing of KPC a. Sub-culture when the cells are approximately 80% confluent.b.Aspirate the medium and wash with 6 mL of PBS.c. Aspirate PBS and add 2 mL of a 0.25 mg/mL trypsin/EDTA solution.Incubate in a 37 °C incubator for approximately 5 min.Check under the microscope if cells are detached.d.Neutralize the enzymatic reaction by adding 4 mL of kidney progenitor cell washing medium and collect the cells in a 15 mL polystyrene tube.Centrifuge the cells at 400× g for 5 min at 4 °C.e. Aspire supernatant without disturbing the cell pellet and resuspend the cells in 5 mL of kidney progenitor cell growth medium.f.Count the number of cells using a Bürker counting chamber.g.Replate the cells in 75 cm 2 flasks with a ratio of 1:3 in kidney progenitor cell growth medium.h.Change medium twice a week during maintenance of cultures in a 5% CO2, 37 °C incubator.

Cryopreservation of KPC
i. Kidney progenitors are cryo-stored in 1 mL of freezing medium at a density from 5 × 10 5 up to 1 × 10 6 cells/cryogenic vial.a. Aspirate the medium from the 75 cm 2 flask and wash with 6 mL of PBS.b.Aspirate the PBS and add 2 mL of a 0.25 mg/mL trypsin/EDTA solution.Incubate in a 37 °C incubator for approximately 5 min.Check under the microscope if cells are detached.c.Neutralize the enzymatic reaction by adding 4 mL of kidney progenitor cell washing medium and collect the cells in a 15 mL polystyrene tube.Centrifuge the cells at 400× g for 5 min at 4 °C.d.Aspire supernatant without disturbing the cell pellet and resuspend the cells in 5 mL of kidney progenitor cell growth medium.e. Count the number of cells using a Bürker counting chamber.f.Centrifuge the cells at 400× g for 5 min at 4 °C.g.Resuspend the cell pellet in freezing medium at a density of 1 × 10 6 cells/mL.Mix well.h.Aliquot in 1 mL per cryovial.i. Transfer the cryovials to a freezing container and put the freezing container into a -80 °C freezer.j.The next day, transfer the cryovials to a liquid nitrogen tank for long-time storage.CAUTION: Minimize as much as possible the time cells remain in freezing medium at room temperature.Transfer immediately in the freezing container to -80 °C.4. Thawing of kidney progenitor cells a. Thaw a vial of cells.To achieve rapid warming, place the frozen vial into a 37 °C water bath.b.Transfer immediately the content of each vial to a 15 mL tube containing 4 mL of kidney progenitor cell washing medium.c.
Centrifuge at 400× g for 5 min at 4 °C.d.Remove the supernatant and resuspend the cells into 2 mL of kidney progenitor cell growth medium.e.
Transfer the cell suspension to 75 cm 2 flasks at a density of approximately 500,000 cells/flask in 8 mL/flask of kidney progenitor cell growth medium.Place the cells in a 5% CO2, 37 °C incubator.

B. Differentiation of KPC into podocytes
1. Detach the cells at 60%-80% confluency with trypsin as described above.
2. Count cells and plate in a 6-well plate at a density of 80,000 cell/well in 1.5 mL/well of kidney progenitor cell growth medium.3. Place the cells in a 5% CO2, 37 °C incubator.4.After 5-6 h (or when cells are attached to the plate), gently remove the medium and replace with 1.5 mL/well of EBM without any supplement and without serum.Place the cells in a 5% CO2, 37 °C incubator.5.After 16 h, remove the EBM medium and stimulate the cells for 48 h with 1.5 mL/well of differentiation medium containing 0.2 μM panobinostat in DMEM-F12 + 10% HyClone Defined FBS. 6.At the end of differentiation, characterize the cells using qRT-PCR and immunofluorescence.CAUTION: To obtain better differentiation results, use cells at early passages (P1-P2).

A. Flow cytometry analysis for purity check
It is important to characterize each primary kidney progenitor cell culture, evaluating in the various passages (from passage P0 to at least passage P3) the expression of surface markers CD133 and CD24.To evaluate CD133 and CD24 expression, perform flow cytometry analysis as reported: 1. Detach the cells with trypsin as described above.
3. Prepare two 1.5 mL tubes: label one Isotype control and the other Antibody (CD133 and CD24).4. Transfer 100,000 cells in each tube.
5. Centrifuge at 400× g for 5 min at 4 °C.6. Aspirate supernatant without disturbing the cell pellet.Add 5 μL of FcR blocking reagent human on cell pellet.7. Prepare the staining solutions as follows: a. Isotype control mix I: Add 1 μL of mouse IgG2b (to obtain a final concentration of 10 μg/mL) and 1 μL of mouse IgG1 (to obtain a final concentration of 10 μg/mL) in 98 μL of FACS buffer.b.Antibody mix I: Add 10 μL of CD133/2 (10 μL/test) antibody and 2 μL of CD24 (to obtain a final concentration of 10 μg/mL) antibody in 28 μL of FACS buffer.8. Add 30 μL of the staining solutions to the corresponding tube containing cells and FcR blocking reagent human.Resuspend the pellet.9. Incubate on ice for 15-30 min covered with a tin foil.10.Add 500 μL of FACS buffer.11.Centrifuge at 400× g for 5 min at 4 °C and aspirate the supernatant without disturbing the cell pellet.12. Resuspend each pellet in 30 μL of the staining solution II, prepared as follows: Staining Solution II: add 1 μL of goat anti-mouse IgG2b-647 (to obtain a final concentration of 5 μg/mL) and 1 μL of goat anti-mouse IgG1-488 (to obtain a final concentration of 5 μg/mL) to 300 μL of FACS buffer.13.Incubate on ice for 15-30 min covered with a tin foil.14.Add 500 μL of FACS buffer.15.Run FACS assay by using MacsQuant Analyzer.16.Analyze the FACS data using Flowlogic software.A representative FACS assay for kidney progenitor cells is shown in Figure 1.CAUTION: Use primary cultures consisting of at least 95% of CD133 and CD24 double-positive cells.The percentage of double-positive cells tend to increase during the first two passages because the kidney progenitor cell growth medium allows the selective growth of undifferentiated kidney progenitors.

B. Evaluation of differentiation
After 48 h of stimuli with differentiation medium, it is possible to evaluate differentiation status of the cells by using qRT-PCR and immunofluorescence (Figure 2).

C. qRT-PCR assay
One of the methods to check the differentiation status of the cells is to use real-time PCR assay to evaluate mRNA expression level of podocyte markers, such as NPHS1, NPHS2, KLF15, and SYNPO.We perform qRT-PCR for each marker on the RNA extracted from an equal number of undifferentiated and differentiated cells independently from the RNA concentration obtained.The qRT-PCR protocol is detailed below: 1. Collect pellets from 100,000 undifferentiated and differentiated cells and extract RNA using the RNeasy Micro kit, followed by the DNase digestion protocol in a final volume of 15 μL per sample.2. Proceed to the synthesis of the cDNA using the TaqMan reverse transcription reagents: a. Prepare the following cDNA synthesis mix on ice in a 0.  .Perform data analysis: gene expression of each marker is normalized to that of the GAPDH.For each marker, results are reported as fold change of expression of the differentiated cells over undifferentiated cells (Figure 2).

D. Immunofluorescence
A differentiative program induces morphology changes and leads to cell-specific protein expression that can be evaluated by immunofluorescence assay.The differentiation of kidney progenitor cells into podocytes is confirmed on the basis of the NPHS1 and NPHS2 podocyte marker expression (Figure 2), while the tubulin and actin expression assessed by super-resolution microscopy shows the drastic cytoskeleton changes associated with differentiation (Figure 3).The immunofluorescence procedure is detailed below: 1. Detach the cells with trypsin as described above.
3. Plate cells onto a 2-well chamber slide at a density of 20,000 cell/well in 1 mL/well of kidney progenitor cell growth medium.4. Place the cells in a 5% CO2, 37 °C incubator.5.After 5-6 h (or when cells are attached to the plate), gently remove the medium and replace with 1.5 mL/well of EBM without any supplement and without serum.Place the cells in the incubator with 5% CO2 and 37 °C.6.After 16 h, remove the EBM medium and stimulate cells for 48 h with 1 mL/well of 0.2 μM panobinostat in DMEM-F12 + 10% HyClone defined FBS. 7. At the end of the differentiation, remove chamber slides from the incubator.8. Aspirate the medium and wash the cells with 500 μL/chamber of PBS.9. Add 1 mL/chamber of 4% PFA and incubate for 20 min at room temperature.10.Gently wash the slides three times with 1 mL of PBS.11.Add permeabilizing solution (composed of 0.5% Triton X-100 in PBS) if required from the antibody user manual (antibody information is reported in Table 2 and Table 3) for 5 min at room temperature.
12. Wash for 5 min with PBS.
13. Incubate with blocking solution containing 3% BSA and 0.3% serum (goat or donkey, as reported in Table 3) in PBS.14. Remove blocking solution without washing.15.Incubate with primary antibody (Table 3) for 15 min at 37 °C and subsequently for 1 h at 4 °C covered with a tin foil.16.Wash for 5 min with PBS.17. Incubate with the secondary antibodies listed in Table 3 and with 1 μg/mL DAPI in PBS 1× for 30 min at room temperature covered with a tin foil to block the light.18. Acquire images using a LEICA SP8 STED 3X confocal microscope.19.For STED analysis, frame sequential acquisition can be applied to avoid fluorescence overlap.A 775 nm pulsed-depletion laser was used and a gating between 0.

Recipes
1. Kidney progenitor cell growth medium (EGM-MV medium + 20% HyClone defined FBS) The renal progenitor cells are grown in EGM-MV medium supplemented with 20% HyClone defined FBS, prepared as follows: Supplement 400 mL of EBM medium with BBE and hEGF provided in the microvascular endothelial growth medium (EGM-MV) SingleQuots kit and with 100 mL of HyClone defined FBS.Filter using a vacuum filtration system with 0.22 μm CA membrane and store at 4 °C.

Kidney progenitor cell washing medium (EBM medium + 10% FBS)
Supplement EBM medium with 10% FBS by using the serum provided with the SingleQuots kit (and not used for EGM-MV + 20% HyClone formulation).Other commercial FBS can be used.Filter the solution using a vacuum filtration system with 0.22 μm CA membrane.

Red blood cell lysis buffer (NH4Cl 0.08%)
Dissolve 0.4 g of NH4Cl in 500 mL of bi-distilled water.Filter using a vacuum filtration system with 0.22 μm CA membrane and store at 4 °C.

Kidney progenitor cell freezing medium
Immediately before freezing the cells, prepare a solution containing HyClone defined FBS supplemented with 10% DMSO.

DMEM-F12 + 10% HyClone defined FBS
Dissolve 15.6 g of powder DMEM-F12 (one vial of DMEM-F12) in 1 L of MilliQ water and supplement with 1.2 g/L NaHCO3.Filter using 0.22 μm filters and store at 4 °C.Add FBS HyClone at a final concentration of 10% (w/v) only for the volume of medium necessary for the experiment.
6. FACS BUFFER (PBS 1× 0.5% BSA -0.02% NaN3 buffer) Dissolve 2 g of NaN3 in 10 mL of PBS to obtain a 20% (w/v) NaN3 stock solution, which can be stored at room temperature for at least two years.Dissolve 1.25 g of BSA in 250 mL of D-PBS, no calcium, no magnesium, and then add 250 μL of 20% NaN3 stock solution.Store at 4 °C.

Figure 1 .
Figure 1.Evaluation of CD133/2 and CD24 expression in kidney progenitor cells by flow cytometry.Representative flow cytometry dot plot graphs showing the percentage of CD133 and CD24 positive cells in primary kidney progenitor cells at passage P1 (B).Staining of the same cells with isotype control antibodies is shown in (A).

Figure 2 .
Figure 2. Evaluation of podocytes derived from kidney progenitor cells.Representative phase contrast images of (A) undifferentiated kidney progenitor cells and (B) podocytes derived from kidney progenitor cells after 48 h of differentiation.Scale bars, 100 μm.(C) qRT-PCR assay of the podocyte markers NPHS1, NPHS2, KLF15, and SYNPO in undifferentiated kidney progenitor cells and in podocytes derived from kidney progenitor cells.mRNA expression of the podocytes markers was determined by qRT-PCR and reported as

10 Published: Aug 20, 2023 7 .
Dispense 15 μL of the mix for NPHS1, NPHS2, KLF15, or SYNPO into each well of a 96-well PCR plate.For GAPDH, dispense 19 μL of the mix.8. Add 5 μL of cDNA samples to the well containing the mix for NPHS1, NPHS2, KLF15, or SYNPO and add 1 μL of cDNA samples to the well containing the mix for GAPDH.9. Seal the plate and centrifuge briefly.Proceed to the incubation below by selecting fast mode: 3 and 6 ns was applied to avoid collection of reflection and autofluorescence.Images were acquired with Leica HC PL APO CS2 100×/1.40oil STED white objective.De-convolve with Huygens Professional software (Romoli et al., 2018).

Figure 3 .
Figure 3. Stimulated emission depletion (STED) super-resolution microscopy shows cytoskeleton changes associated with differentiation.Representative STED images showing cytoskeleton changes associated with